Our laboratory has for a number of years been interested in the structure and function of membrane-bound enzymes. During the past several years we have purified the two cytochrome complexes which are the membrane-bound terminal oxidases of the aerobic respiratory chain of E. coli. One of these enzymes, the cytochrome d terminal oxidase complex, is the focus of all the work proposed in this grant. The cyto-chrome d complex is of particular importance in that it is representative of a class of membrane-enzymes which couples an electron transfer reaction to the electrogenic translocation of protons across the membrane bilayer. Our long term goal is to understand the mechanism by which this enzyme generates a proton motive force. Many diseases are characterized by bioenergetic deficiencies, and understanding the structure and mechanism of a protein which functions as a "coupling site" is of fundamental importance in elucidating the nature of some of these defects. In order to accomplish our goal, we are emphasizing the use of molecular genetics and immunological techniques directed at obtaining structural information about this protein. We will utilize several approaches to determine which portions of each polypeptide are periplasmic, cytoplasmic, or buried with the membrane bilayer. Proteolysis, antibodies binding to defined epitopes, and gene-fusion technology will all be used to define the protein topography within the cytoplasmic membrane. Specific amino acid residues involved in catalysis or heme-binding will be identified primarily through the use of genetics. In particular, site-directed mutagenesis will be used to define which of the total of 10 histidine residues act as heme axial ligands. Biophysical and biochemical approaches will also be used to obtain similar information and help clarify the specific role played by each of the heme centers in this enzyme. Finally, the genetic expression of this enzyme is regulated by oxygen, and we will identify the genes required for this regulation as well as the specific DNA sequence in the cytochrome operon (cyd) which is responsible for the response to oxygen.